The species of Phyllanthus have been used since ancient times in different systems of medicine, particularly for the treatment of liver disorders and urinary infections but the concepts and identification of various species, particularly herbaceous ones, have been unclear mainly due to misidentification of specimens in several herbaria. The literature survey and extensive field studies conducted by Chaudhary and Rao (2002), Phytotaxonomy 2: 143-162 revealed that out of the 40 species occurring in India, chiefly the allied herbaceous species, like P. amarus, P. fraternus, P. debilis are extensively used for jaundice and hepatitis whereas P. urinaria for urinary tract infection. These species are also used in stomach ailments like dyspepsia, colic, diarrhea, dysentery, dropsy, urinogenital problems and also as external application for oedomatous swelling and inflammation. These species are important ingredient in many ayurvedic preparations, especially for the treatment of jaundice. This species Phyllanthus amarus is distributed throughout India and is considered to be the most widely occurring species of Phyllanthus in India.
In recent years, the interest in the plants of the genus Phyllanthus has increased considerably due to its therapeutic potential for the treatment of different diseases. Substantial literature on the chemistry and the pharmacological and clinical properties of the genus is available. Different classes of compounds have been isolated from the plants of this genus. Most of these compounds were found to interact with key enzymes of the animal/human system. But most of the time the species are confused or not properly identified leading to adulteration of crude drugs. The genus Phyllanthus niruri is non-existent in India. Hence the research works in the name of P. niruri is either similar species like Phyllanthus amarus, Phyllanthus fraternus or Phyllanthus debilis; or abundantly available species of Phyllanthus urinaria. So it is necessary to develop a marker system by which the individual species can be differentiated and more specifically the presence of P. amarus in the crude drug can be ascertained. Hence systematic experimentation was undertaken to develop a marker system for P. amarus, P. fraternus, P. debilis and P. urinaria. 
Many genetic-marker technologies are adaptable to fingerprinting, including restriction-fragment-length polymorphism (RFLP) Bostein et al. (1980) Am J Hum Genet 32:314-331; single strand conformation polymorphism (SSCP) Fischer et al. (1983) Proc Natl Acad Sci USA 80:1579-1583, Orita et al. (1989) Genomics 5:874-879; amplified fragment-length polymorphism (AFLP) Vos et al. (1995) Nucleic Acids Res 23:4407-4414; microsatellite or single-sequence repeat (SSR) Weber J L and May P E (1989) Am J Hum Genet 44:388-396; rapid-amplified polymorphic DNA (RAPD) Williams et al. (1990) Nucleic Acids Res 18:6531-6535; sequence tagged site (STS) Olson et al. (1989) Science 245:1434-1435; genetic-bit analysis (GBA) Nikiforov et al. (1994) Nucleic Acids Res 22:4167-4175; allele-specific polymerase chain reaction (ASPCR) Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448, Newton et al. (1989) Nucleic Acids Res 17:2503-2516; nick-translation PCR (e.g., TaqMan™) Lee et al. (1993) Nucleic Acids Res 21:3761-3766 and allele-specific hybridization (ASH) Wallace et al. (1979) Nucleic Acids Res 6:3543-3557, (Sheldon et al. (1993) Clinical Chemistry 39(4):718-719) among others. Kits for RAPD and AFLP analyses are commercially available, e.g., from Perkin Elmer Applied Biosystems (Foster City, Calif.). For example, the restriction fragment length polymorphism (RFLP) technique employs restriction enzyme digestion of DNA, followed by size separation of the digested DNA by gel electrophoresis, and hybridization of the size-separated DNA with a specific polynucleotide fragment. Differences in the size of the restriction fragments to which the polynucleotide probe binds reflect sequence differences in DNA samples, or DNA polymorphisms. See Tanksley, Biotechnology 7:257-264 (1988). (McCasky Feazel, et al. 2000. U.S. Pat. No. 6,100,030); (Jain, N et al., Current Science. 85 (10):1454-1458 (2003), (Hosokawa K et al Planta Med. 66(3): 270-2 (2000)).
The novelty of the present invention is the unique DNA sequences those identified by using the simple Randomly amplified polymorphic DNA profile analysis and the methodology by which the SCAR markers can be used to detect or confirm the presence of different species in a crude drug mixture as it is not possible to identify the species in a crude drug morphologically. Also chemical analysis may not be reliable as a species may not produce all the chemicals at all developmental stages. Further the genotypes of plants within the species differ leading to differences in the chemical profiles. Till date full proof commercial methods to identify the species in the mixture of crude drug are not available. It is difficult to identify the investigated species in the intact plants itself and nearly impossible to identify the species in crude drug through conventional methods. So the present invention will help in determining the quality of crude drug with confirmation through these assigned DNA markers.
Mainly the herbaceous species like P. amarus Schum. and Thonn, P. fraternus Webster and P. debilis Klein ex Willd have been reported to be extensively used for jaundice and P. urinaria L for urinary tract diseases. Modern research with Phyllanthus focuses on its potential for fighting viruses, specifically the hepatitis B virus (Shin et al. Antiviral Research, 67,163-168 (2005)) and malaria parasite (Hout et al. Journal of Ethnopharmacology, 107, 12-18 (2006)). Despite extensive use for a long time, confusion still prevails over the correct identity and nomenclature of the species as it commonly known as “Bhuianavala” or “Bhumyamalaki” in Indian literature and Ayurveda, representing a group of species comprising of P. amarus, P. fraternus, P. debilis and even P. urinaria (FIG. 1). Several earlier workers included all these species especially P. amarus, P. fraternus and P. debilis under a single species P. niruri which was later described as ‘niruri complex’. The species P. niruri is an American species and not at all found in India. Webster (Journal of the Arnold Arboretum, 38,295-373 (1957)) and Mitra and Jain (Bulletin of the Botanical Survey of India, 27, 161-176 (1985)) showed that the P. niruri of Hooker is actually represented by above described three different species. Thus, confusion in identification of these herbaceous species is largely due to common vernacular names for all the species, similarity in gross morphology, close proximity in the growth habitat and extent of diverse morphological features of the species. This misidentification leads to deliberate or ignorant adulteration of crude drugs, lowering the efficiency of the medication for which it has to be used specifically, leading to decreased or deleterious effect of the drug when administered. So the present study was targeted to develop molecular markers, which would help in unambiguous identification of the above species. The marker can also be used in quality check in dry herbs samples exported from India.